Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

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Abstract

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F619 in the motif stacks partly over cis-Pro620 in one conformation, whereas it is swung out from cis-P620, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.

Highlights

► We determined the crystal structure of human FAF1 UBX domain. ► The structure reveals a novel FcisP touch-turn motif in p97-binding region. ► The structure presents two different conformations of p97-binding region. ► The two conformations should represent the unbound and bound states.

Introduction

p97/VCP, a multi-functional ATPase of the AAA+ family, is implicated in diverse cellular processes such as post-mitotic Golgi reassembly, endoplasmic reticulum associated degradation, nuclear envelope reconstruction, cell cycle, suppression of apoptosis and DNA damage response [1]. The diverse functions of p97/VCP are mediated by the interaction with various adaptor proteins in each different biological context [2], [3]. The largest group of the adaptors comprises the proteins containing the UBX domain, such as FAF1, p47, SAKS1, and UBXD7, which directly binds to N domain of p97/VCP [4], [5], [6], [7]. The UBX domain comprises about 80 amino acid residues and is currently considered as a general p97/VCP-binding module, with a growing number of UBX proteins being identified [8]. The structures of UBX domain were determined by NMR spectroscopy on FAF1 UBX and p47 UBX, revealing that it adopts a β-grasp fold similar to ubiquitin [5], [9]. The first structural glimpse on its interaction with p97/VCP was obtained from the crystal structure of p47 UBX in complex with p97/VCP ND1 fragment at 2.9 Å resolution [10]. It showed that the conserved FP sequence motif in the loop connecting strands S3 and S4 is inserted into the cleft of the p97/VCP N domain and is essential for the binding activity [10].

FAF1 was initially identified as a Fas-interacting protein in the death inducing signaling complex and was shown to potentiate the apoptotic cell death [11], [12], [13]. Later, FAF1 was shown to intervene in the NF-κB signaling at two points of the pathway. First, FAF1 binds to the IKK β subunit, thereby disrupting the IKK complex assembly and inhibiting its activation [14]. Second, FAF1 also binds to p65 subunit of NF-κB and inhibits the translocation of NF-κB into the nucleus [15]. Most recently, FAF1 was revealed to inhibit the proteasome-mediated protein degradation process either by interacting with p97/VCP which may serve as a molecular chaperone presenting the ubiquitinated client proteins to the proteasome or by interacting with ubiquitinated client proteins [4]. As a consequence of its involvement in these cellular processes, FAF1 acts as a pro-apoptotic protein.

Human FAF1, composed of 650 amino acid residues, contains a UBX domain at the C-terminus through which it binds to p97/VCP N domain [4], [16]. In an effort to elucidate the conserved structural features of the UBX domain for its binding to p97/VCP and also to provide a structural basis of the interaction between FAF1 and p97/VCP, we initiated X-ray crystallographic analysis on FAF1 UBX domain, and here we present its crystal structure determined at 2.9 Å resolution.

Section snippets

Protein preparation, crystallization and data collection

The human FAF1 UBX domain (residues 571–650) was overexpressed, purified and crystallized as described previously [17]. Briefly, FAF1 UBX protein was overexpressed in fusion with His-tagged thioredoxin in Escherichia coli strain Rosseta2 (DE3). The fusion part was removed by TEV protease during the purification process. The FAF1 UBX alone was concentrated to 9.7 mg/ml for its crystallization. We collected X-ray diffraction data to 2.9 Å resolution, at Photon Factory, Japan on the beamlines

Overall structure of FAF1 UBX

FAF1 UBX domain comprises five β-strands (S1–S5), one α-helix (H1), and three 310 helices (G1–G3) (Fig. 1A). The five strands form a mixed β-sheet in the order of 2–1–5–3–4, and the sheet rolls around the α-helix H1 to form the well-known β-grasp fold, as previously shown in the NMR structure of FAF1 UBX [9]. Notably, among the three short 310 helices G1 to G3, only G3 was recognized in the previous NMR structure as a helix H2.

FAF1 UBX domain contains three Phe-Pro sequence stretches: FP608-9,

Acknowledgments

This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080930). The authors thank the staff of the Pohang Light Source beamlines 4A in Korea, and the Photon Factory beamlines AR-NW12 in Japan.

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